The past two decades have provided major advances in understanding of the genetics, physiology and biochemistry of cystic fibrosis. However, as yet this information has not been fully exploited to provide new molecular therapies that benefit the patients. Although the identification of the CFTR gene and disease associated mutations has enabled DNA-based screening which is now the standard of care, progress towards gene replacement therapy has not proceeded as rapidly as anticipated. Therefore alternative approaches to the development of new treatments are required. Despite the many complexities and challenging aspects of the disease there is at least one feature that provides an opportunity for manipulation at the molecular level. Approximately 90% of patients have a mutation, deltaF508 that allows a potentially functional CFTR protein to be synthesized. The protein is detected as abnormal by endoplasmic reticulum quality control and prevented from trafficking to the apical plasma membrane of epithelial cells where its chloride channel activity is required. However, this mutation is temperature sensitive and its effects can be circumvented in cells grown at reduced temperature or by protein stabilizing agents such as some ampholytes. Hence the search for small molecule drugs that could have these effects becomes an attractive strategy which already has been taken by two other groups that have initiated high throughput screening (HTS) efforts using assays that indirectly measure restoration of deltaF508 CFTR chloride channel activity with fluorescent probes that sense changes in membrane potential or chloride concentration. These assays are useful and one has already identified new modulators of CFTR channel activity but not yet agents that overcome AF508 misprocessing. However many changes other than mutant CFTR maturation can give signals in these indirect readouts (false positives). Therefore we have initiated development of an HTS that directly measures the appearance and stability of the deltaF508 protein at the cell surface by insertion of an exogenous epitome into a modified extracytoplasmic loop of CFTR without perturbing its synthesis, glycosylation or function. Our specific objectives in this proposal are to further develop and optimize this assay as a highly sensitive luminescence cell-based HTS to provide stringent validation assays and to initiate screening of a diverse library of compounds in collaboration with a Molecular Libraries Screening Center.